Summary/Abstract: Pertussis (aka whooping cough) is a serious reemerging public health problem with incidence estimated at 20 million cases annually and deaths (mostly in infants) at ~200,000 annually. Recent rises in pertussis in countries with high vaccine coverage, such as the United States, correlate with a switch from whole cell (wP) to acellular (aP) pertussis vaccines and are attributed to a larger reservoir of infected individuals composed of adolescents and young adults who were vaccinated (rather than infected) as children. It is now apparent that immunity induced by aP vaccination is not as durable as vaccination by wP vaccination, which is not as durable as immunity induced by infection with Bordetella pertussis, the primary causal agent of pertussis. Moreover, while immunization with wP and aP vaccines provides protection against disease (at least initially), it does not protect against colonization. New vaccines that provide sterilizing, long-lasting immunity are needed. We have discovered a previously uncharacterized signal transduction system (PlrSR) that is required for B. pertussis and the closely-related broad host range pathogen Bordetella bronchiseptica to colonize and persist in the lower respiratory tract (LRT). Our preliminary data support a model in which PlrSR functions as a kinase in response to increased CO2 and low oxygen conditions (reflective of the LRT), resulting in high levels of PlrR-phosphate (PlrR~P) that activate expression of genes including those encoding high-affinity cytochrome oxidases. Our model states that under aerobic conditions, PlrS functions primarily as a phosphatase, and that low levels of PlrR~P are essential for cell viability. We will use genetic, molecular biological, and genome-wide approaches to identify PlrSR-regulated genes, especially those induced only in the LRT, and will determine the roles of PlrSR-dependent gene regulation and of the factors encoded by the regulated genes in virulence. Using genetic approaches, we will determine the role of the PlrS PDC and PAS domains, as well as the predicted kinase and phosphatase activities of PlrS, in the ability of the bacteria to grow in vitro and in the LRT. Using biochemical approaches, we will determine if PlrS is a redox- sensitive heme-containing protein that functions as a kinase under low oxygen conditions and a phosphatase in ambient air, and we will identify DNA sequences to which PlrR~P binds. Our results will be significant because previously unknown PlrSR-dependent virulence factors, and the PlrSR system itself, will be excellent candidates for the development of new component vaccines and targets for the development of new therapeutics. Our results will also advance our understanding NtrYX family proteins (of which PlrSR is a member and which control virulence in other pathogens) function and they will provide insight into how respiratory pathogens, in general, grow in the LRT.